DE69520725T3 - Method for the diagnosis of blood coagulation disorders - Google Patents

Abstract

A method and kit for screening blood plasma for APC cofactor is provided. The method comprises: (i) incubating a first portion of the sample with (a) an exogenous reagent containing phospholipid which activates the contact mechanism in intrinsic clotting or an exogenous reagent which initiates extrinsic clotting; and incubating a second portion of the sample with (b) the reagent of step (i) (a) and an exogenous Protein C (PC) activator which produces activated PC (APC) from endogenous Protein C and Protein S. (ii) adding to each of the two portions a clotting agent and monitoring the conversion rate of fibrinogen to insoluble fibrin; and (iii) calculating the ratio of the rate for the second portion of the sample to the rate for the first portion and comparing this ratio with the corresponding ratio for samples from normal subjects.

Description

The The present invention relates to a novel process, which relates to Screening and diagnosis of thromboembolic diseases, e.g. the hereditary thrombophilia is of use. The invention can also used to reduce the risk of thrombosis in pregnant women, Patients undergoing surgery and persons the contraceptives take, determine.

The Blood clotting involves a complex system of linked proenzymes, Enzymes and cofactors that are on the surface of activated platelets and Endothelial cells react. From an activation of the system follows Ultimately the formation of a blood clot, the insoluble Contains fibrin. The beginning and end of fibrin formation are carefully regulated during normal homeostasis. In vitro, platelet and endothelial cell surfaces usually replaced by suitable phospholipids. The relevant part of the invention of the coagulation system is shown below.

protein C is a zymogen produced in vitro by thrombin (factor IIa) alone, the combination of thrombin thrombomodulin, a fraction of certain snake poisons, such as Agkistrodon Contortrix Contortrix, or purified factor Xa can be activated. By the activation of endogenous protein C of a plasma sample or the Add exogenously activated protein C to a plasma sample in the presence of protein S (PS) and activated protein C (APC) cofactor the factors Va and VIIIa neutralized and blood coagulation times prolonged in the plasma samples of healthy persons. Factors V and VIII are activated by small amounts of thrombin or factor Xa. Protein S is a cofactor to protein C. A hereditary homozygous or heterozygous deficiency of protein C, protein S and antithrombin III (ATIII, an inhibitor of factors XIa, IXa, Xa and thrombin) is about 10-15% of patients diagnosed with thromboembolic disease to find under the age of 40 years. A homozygous protein C and -S deficiency is life-threatening; developed in affected individuals a general microvascular thrombosis and purpura fulminans in the neonatal period.

One Lack of protein C and S and ATIII is based on functional and immunological method. A method of determination Functional protein C activity includes the following Steps: Mixing the plasma sample to be tested with an excess on human plasma with protein C deficiency, adding a protein C activator and observing the corresponding substrate conversion. As an alternative, substrates for enzymes such as thrombin and factor Xa used; their activity is going through affects activated protein C activity (i.e., enzyme activities that generated or modulated by APC). In certain cases have Protein C tests the isolation of the zymogen and subsequent activation and addition from substrate to activated form.

For determining the functional protein S activity in a plasma sample, the most common methods include mixing the plasma sample with activated protein C, an excess of plasma with prote In-S deficiency and other reagents necessary to achieve coagulation (Waart et al., Thromb. Res. 48, 427-37 (1987); Suzuki et al., Thromb. Res. 49, 241-51) (1988); Bertina et al., Thromb. Haemost. 53, 268-72 (1985); and Comp et al., J. Clin. Invest. 74, 2084-88 (1984)). It has also been proposed to measure protein S by incubating the plasma sample with factor IXa and activated protein C and measuring the clotting time or the conversion of a chromogenic thrombin substrate (WO-A-9101382).

In addition to Protein C and protein S abnormalities were shown (Dahlback B, Carlsson M and Svensson BJ. Proc. Natl. Acad. Sci. 1993; 90: 1004-08) that the most frequent abnormality hereditary thrombophilia associated with the APC cofactor is.

This cofactor is part of the Accelerator Factor V molecule, with which APC forms an inhibitor that destroys Factor Va and Factor VIIIa, thereby regulating the coagulation mechanism and preventing thrombosis. The abnormality in the Factor V molecule, which renders susceptible to thrombosis, is usually the result of a transient mutation leading to a substitution of Arg ⁵⁰⁶ by Gln ⁵⁰⁶ , known as Factor V Leiden.

• Eine von einem Versuchsobjekt erhaltene Plasmaprobe wurde 5 Minuten lang inkubiert mit
• (a) einem exogenen Reagens (1), das den intrinsischen Gerinnungsmechanismus aktiviert, gefolgt von der Zugabe von Calciumchlorid (2), um die Koagulationsreaktion abzuschließen; und separat mit
• (b) dem gleichen exogenen Reagens (1), in dem die Koagulationsreaktion durch die Zugabe von Calciumchlorid abgeschlossen wurde, das ein exogenes APC-Präparat (3) enthielt.

In the aforementioned document by Dahlback et al., A specific in vitro assay for the detection of the APC cofactor in human plasma samples has been described as follows:

• A plasma sample obtained from a test subject was incubated for 5 minutes with
• (a) an exogenous reagent (1) which activates the intrinsic coagulation mechanism, followed by the addition of calcium chloride (2) to complete the coagulation reaction; and separately with
• (b) the same exogenous reagent (1) in which the coagulation reaction was completed by the addition of calcium chloride containing an exogenous APC preparation (3).

step (a) is considered an ordinary one activated partial thromboplastin time (APTT) known. step (b) is an APTT with addition of APC. The latter bypasses the mechanism of inhibition in conjunction with protein C and protein S. The clotting times a sample in step (a) will vary depending on the type of exogenous activating agent (APTT reagent) between 25 seconds and 45 seconds. In attendance of a normal APC cofactor, the clotting time in step (b) increased. Dahlback pressed this as a b / a ratio from, i. as APC.APTT / APTT ratio. Usually this ratio is between 2.0 and 4.0, in patients with heterozygous or homozygous APC cofactor defects is the ratio below 2.0. To diagnose the various defects in thrombophilia in conjunction with protein C need Therefore, three separate tests are carried out: a protein C test, a protein S test and an APC cofactor test. With the APC cofactor test alone, the presence or absence of PS and PC is not detected.

For a correct Measurement of the APC.RPTT / APTT ratio in patients who are treated for thrombophilia will beat e.g. Jorquera et al, Lancet 1994, Vol. 344, pp. 1162-3, the plasma sample in a ratio of 1: 5 in Factor V-depleted plasma.

Protein C can be activated by a fraction of the snake venom A. Contortrix Contortrix, known as protein C activator (PCA), and if PCA is added to normal plasma, then the inhibition mechanism becomes associated with PC, PS and APC Cofactor triggered by endogenous / endogenous PC, PS and APC cofactor. WO 94/17415 discloses a test for APC activity which is similar to the APC cofactor assay described above in the Dahlback et al document, except that a PCA-calcium chloride mixture is added instead of an APC-calcium chloride mixture to complete the coagulation reaction. The EP 0 236 985 discloses a spectrophotometric method for determining protein C or protein S activity in a plasma sample based on measurements of the amount of thrombin produced upon activation of endogenous protein C with an exogenous PCA.

The The present invention provides a test method for determining the Presence or absence of APC cofactor in human plasma samples ready, the according to a Examination of normal PC and PS values. Be with him Furthermore homozygous PC and PS defects and most, but not all, heterozygous PC defects detected. For the samples examined it is human plasma.

The The invention provides an in vitro screening method for the exclusion of APC cofactor disorders as the cause of thrombophilia in a subject, e.g. one Subject or to determine the risk of acquiring Thrombophilia ready for a test object.

According to the invention, an APC cofactor fishing test is provided for determining whether a blood Plasma sample in which the values of protein C and protein S are normal after assay, has a lack of APC cofactor comprising:

• (a) einem exogenen Reagens, das Phospholipid enthält, das den Kontaktmechanismus beim intrinsischen Gerinnen aktiviert (ein APTT-Reagens), oder einem exogenen Reagens, das die extrinsische Gerinnung auslöst (ein Prothrombinzeit-(PT)-Reagens); und Inkubieren eines zweiten Teils der Probe mit
• (b) dem Reagens aus Schritt (i)(a) und einem exogenen Protein-C-(PC)-Aktivator, der aktiviertes PC (APC) von endogenem Protein C und Protein S produziert;
• anschließend (ii) Hinzufügen eines Gerinnungsmittels zu jedem der zwei inkubierten Teile, wie z.B. ein zweiwertiges oder dreiwertiges Salz, vorzugsweise Calciumchlorid, und Überwachen der Umwandlungsgeschwindigkeit von Fibrinogen in unlösliches Fibrin, zum Beispiel manuell oder mit einem mechanischen, photoelektrischen oder nephelometrischen Instrument; und
• (iii) Berechnen des Verhältnisses zwischen der Geschwindigkeit beim zweiten Teil der Probe und der Geschwindigkeit beim ersten Teil und Vergleichen dieses Verhältnisses mit dem entsprechenden Verhältnis bei Proben von normalen Versuchsobjekten.

(i) incubating a first portion of the sample at preferably 37 ° C

• (a) an exogenous reagent containing phospholipid that activates the intrinsic channeling mechanism of contact (an APTT reagent) or an exogenous reagent that induces extrinsic coagulation (a prothrombin time (PT) reagent); and incubating a second portion of the sample with
• (b) the reagent of step (i) (a) and an exogenous protein C (PC) activator producing activated PC (APC) of endogenous protein C and protein S;
• then (ii) adding a coagulant to each of the two incubated parts, such as a divalent or trivalent salt, preferably calcium chloride, and monitoring the rate of conversion of fibrinogen to insoluble fibrin, for example, manually or with a mechanical, photoelectric or nephelometric instrument; and
• (iii) calculating the ratio between the velocity at the second part of the sample and the velocity at the first part and comparing that ratio with the corresponding ratio for samples from normal test objects.

The relationship is called PCA.APTT / APTT ratio or as PCA.PT/PT ratio designated.

The Invention is an in vitro method used as a screening test method is applied to APC cofactor defects ruled out which lead to thrombophilia could. APC cofactor defects are likely to occur in less than 5% of cases normal population on. It is believed that the method of the invention is more discriminatory as a test method of the prior art, such as the APC cofactor test, having.

We have found that the PCA-APTT ratio in normal individuals is over 2.0 and even 10.0, whereas PCA.APTT ratios in APC cofactor defects between 2.0 and 1.0. These numbers can be used for different APTT reagents and different instruments vary slightly, with the lower limit of normality at a ratio can be between 2.2 and 2.5.

The Invention also provides a clearer test for APC resistance ready. A potential problem in connection with the APC cofactor test is that patients with thrombophilia be subjected to an anticoagulant treatment, the leads to a reduction of factors II, IX and X. This has the consequence that the above-described APC cofactor test has a higher PCA.APTT / APTT or PCA.PT/PT ratio, as it should be, what a potential misdiagnosis of APC-resistant patients may result as normal. According to this second aspect of the invention, the factors II, IX and X in the plasma sample (which may be APC-resistant or sensitive) normalized by diluting with Factor V-depleted plasma. Furthermore, a dilution normalizes in factor V, the values of PC and PS in the diluted sample, regardless of the values in the undiluted Sample. Preferably, the plasma is in a ratio of 1: 5 or 1:10 in the Factor V-depleted Diluted plasma. The PCA.APTT / APTT or PCA.PT/PT ratio is then determined according to the above described general aspect of the invention.

APC cofactor and factor V coagulant activity are both part of the Factor V molecule and therefore not factor-V depleted Plasma available. The dilution holds therefore the balance in the plasma sample between the coagulant factor V and the anticoagulant APC cofactor upright.

The intrinsic coagulation factors and PC and PS are located in any diluted sample at about the same level; hence the range of PCA.APTT / APTT or PCA.PT / PT values observed in plasma samples from normal patients becomes, narrower as the corresponding area in testing using of undiluted Plasma.

The The exogenous APTT reagent used in step (i) (a) may be micronized Silica (preferably at about 0.06% in water and buffer), kaolin (preferably between 0.25 and 10 mg / ml), bentonite, ellagic acid or contain any substance that makes the contact mechanism intrinsic Clotting activated, or any commercial liquid or lyophilized APTT reagent. The Phospholipid may be a platelet substitute like Brain extract or a relatively pure phospholipid.

The exogenous PT reagent used in step (i) (a) in conjunction with the extrinsic pathway may be bovine brain thromboplastin or any other organ species having a longer coagulation time at norma plasma in the presence of PCA. Preferably, the PT reagent is the supernatant following centrifugation (preferably about 500 g) of a 4% solution of acetone-dried bovine brain activated in saline. Brain thromboplastin from humans or rabbits is unsatisfactory.

The protein C activator used in step (i) (b) may be derived from human or animal components or fractions of snake venoms or any other protein C exogenous activator. The PCA used in step (i) (b) is combined with exogenous activator either in liquid or lyophilized form. We have used a fraction from A. Contortrix Contortrix (Exner T and Vaasjcki R. Thromb, Haemost., 59: 40-44 (1988)). The concentration of the PC activator is preferably between 0.25 × 10 ^-8 M and 2.5 × 10 ^-8 M, with 1.25 × 10 ^-8 M being most preferred.

The optimal dilution in the APTT or PT reagent is that which is the maximum extension the clotting time of normal pooled plasma in step (ii) for the product achieved from step (i) (b). It can higher or higher lower concentrations are used, however, in both cases the clotting times are shorter, the PCA ratios less and the results less sensitive.

Preferably The reagents are in lyophilized form and are with Water or another diluent reconstituted.

The Calcium chloride from step (ii) is optionally 25 mM, but it can also concentrations between 10 mM and 50 mM can be used. Concentrations over 25mM may have an inhibitory effect, and concentrations below 10 mM are sufficient for coagulation possibly not from. It can Magnesium and other divalent and trivalent cations are involved become.

in the The state of the art becomes saline used to prepare the portion of the sample from which the APC.APTT time is measured. It is preferred to use saline both in the APTT and also to use in the PCA.APTT measurement to ensure that the observed clotting times are properly comparable. Preferably, the concentration of saline is between 0.075 M and 0.15 M.

The contains exogenous reagent preferably between 0.01 and 0.2%, preferably 0.04, of crude phospholipid. The optimal incubation time in step (i) is 5 to 10 minutes for the APTT and the PT reagent, it can but also periods of only 2 minutes or up to 15 minutes. Longer incubation times can lead to a deterioration of the components in the plasma sample.

According to the present Invention will also a kit to carry out the method according to the invention comprising an APTT reagent or a PT reagent and, separately, the same reagent with a PC activator. Furthermore, the kit comprises a coagulant, preferably a divalent or trivalent salt, with calcium chloride on the most preferred. The kit preferably contains sodium chloride, which can be combined with the coagulation activator. The concentration the sodium chloride solution is preferably between 0.075 M and 0.15 M.

EXAMPLE

A APTT with and without inclusion of a PCA reagent.

Method: Citrated blood is preferred, but oxalate or EDTA may also be used Anticoagulant can be used. Plasma is made by centrifugation obtained at 3000 g. We have the test manually and in APTT mode with the ACL instrument from Instrumentation Laboratory, Milan, Italy, performed. It can also other commercial ones Instruments are used. The manual procedure will be described.

Method (i): Plasma (0.1 ml) is placed in a coagulation tube at 37 ° C, followed by APTT reagent (Diagen micronized silica APTT reagent, Thame, Oxon, UK) (0.1 ml) The content becomes mixed and incubated for 5 minutes. Subsequently, a mixture of 1 part of 50 mM calcium chloride and 1 part of physiological (0.15 M) saline (0.1 ml) is added and the clotting time is determined by means of the tilt-tube method. This is the normal APTT; Normal clotting times are between 30 and 45 seconds. The assay procedure is repeated with the same APTT reagent containing PCA at 1.25 x 10 ^-8 M (Diagen-PCA.APTT reagent). The normal clotting time is between 65 and 300 seconds and gives a ratio between 2.0 and 10.0 when divided by the normal APTT.

Method (ii): Plasma (0.1 ml) is placed in a coagulation tube at 37 ° C, followed by bovine thromboplastin (Diagen-bovine thromboplastin, Thame, Oxon, UK) (0.1 ml) The contents are mixed and incubated for 5 minutes. Subsequently, 25 mM calcium chloride (0.1 ml) is added and the clotting time is determined by the flip-flop method. This is the normal prothrombin time (PT); Normal clotting times are between 27 and 42 sec. The assay procedure is repeated with the same bovine thromboplastin reagent containing PCA at 2.5 x 10 ^-8 M (Diagen Bovine Thromboplastin PCA Reagent). With PCA, the clotting time is between 45 and 60 seconds, yielding a ratio between 1.6 and 2.5 when divided by the normal PT.

The Method (i) is due to the greater increase in clotting times in the presence of PCA preferred. This will increase the sensitivity in the proof of abnormality achieved.

TEST RESULTS

plasma samples of four patients with known APC cofactor defects, 57 normal plasma samples and 58 consecutive plasma samples from patients undergoing thrombophilia screening were tested with the PCA.APTT test and the APC cofactor test Chromogenix (Quadratech, Epsom, Surrey, UK). The border the abnormal range was 2.1 in both tests or less. All four known APC cofactor defects resulted in both test procedures ratios less than 2.1. Of the 57 normal plasma samples three in both test ratios of less than 2.1, indicating a defect in the APC cofactor. Of the 58 patients which were screened for thrombophilia had three ratios of less than 2.1 in both test procedures, four indicated ratios less than 2.0 in the PCA-RPTT test, but in the APC.APTT test ratios of 2.1, 2.2, 2.4 and 2.6. All four had a normal PC. It was assumed that these four samples may be heterozygous in Regarding the APC cofactor defect are; genomic DNA from three of these patients was subjected to polymerase chain reaction amplification, which revealed that they were heterozygous for factor V Leiden mutation. One patient sample had a ratio of 2.5 in the APC.APTT test but an abnormal PCA-APTT ratio of 1.7. A specific one PC examination revealed that the sample only 29% of the average normal PC had. All other abnormal samples were within limits the investigation a normal PC and PS on. A sample, immuno-compromised in PS [* 2], yielded an APC.APTT ratio of 1.7 and a PCA.APTT ratio of only 1.3. It was thus shown that the PCA method all APC cofactor defects proved by the APC method and also PC and PS. Normal samples generally had higher ratios PCA procedures, and seven out of nine abnormal samples yielded lower ratios in the PCA process. Along with the detection of four additional abnormal samples reflected this may be easy a greater discrimination of the PCA test procedure. The test would have to be considered in routine hospital procedures a useful one APC resistance test. The use of the PCA.APTT test can also in discovering other possible factors or cofactors be of use, which participates in the endogenous formation of APC could be.

In Method according to the second Aspect of the invention when the patient is treated for thrombophilia or can be, corresponds to the methodology of the general test procedure, however, the plasma sample is initially in a ratio of Diluted 1: 5 to 1:10 in Factor V-depleted plasma. The PCA.APTT / APTT or PCA.PT/PT ratio then as with undiluted Plasma determined; the results reliably indicate an APC cofactor deficiency. By doing previous example would the 0.1 ml plasma sample therefore 20 μl patient plasma and 80 μl factor V depleted Plasma include.

Claims (18)

APC Cofactor Failure Test for determining whether a blood plasma sample in which the values of protein C and protein S are normal after assay has a deficiency of APC cofactor, comprising the steps of: (i) incubating a first portion of the sample with (a ) an exogenous reagent containing phospholipid, which activates the intrinsic coagulation contact mechanism, or an exogenous reagent which induces extrinsic coagulation; and incubating a second portion of the sample with (b) the reagent of step (i) (a) and an exogenous protein C (PC) activator producing activated PC (APC) of endogenous protein C and protein S; then (ii) adding a coagulant to each of the two incubated parts and monitoring the rate of conversion of fibrinogen to insoluble fibrin; and (iii) calculating the ratio between the velocity at the second part of the sample and the Ge speed at the first part and comparing this ratio with the corresponding ratio for samples from normal experimental subjects. APC cofactor fishing test comprising the steps of (i) to (iii), wherein the plasma sample is initially with factor V-depleted plasma dilute becomes. Test according to claim 2, wherein the dilution of the Plasma sample in factor-V depleted plasma is between 1: 5 and 1:10. Test according to one of the preceding claims, wherein the coagulant added in step (ii) is a bivalent one or trivalent salt, and preferably calcium chloride. Test according to one of the preceding claims, wherein the parts are incubated with an exogenous reagent, with sodium chloride into the products of step (i) (a) and (i) (b). The test of claim 5, wherein the saline solution concentration in the products between 0.075 M and 0.15 M. Test according to one of the preceding claims, wherein the reagent of step (i) is (a) an APTT reagent. Test according to one of the preceding claims, wherein the conversion rate observed in step (ii) the clot formation is observed. Test according to one of the preceding claims, wherein the exogenous protein C activator any exogenous activator of Protein C or protein S is. Test according to one of the preceding claims, wherein the reagents are in lyophilized form and with water or another diluent be reconstituted. Kit for use in a test according to one of previous claims, full: an exogenous reagent containing phospholipid, the activates the contact mechanism in intrinsic coagulation, or an exogenous reagent that triggers extrinsic coagulation; and, separately, the same exogenous reagent with an exogenous protein C (PC) activator, the activated PC (APC) produces endogenous protein C and protein S; and a coagulant, which is preferably a bivalent or trivalent salt, with calcium chloride being most preferred becomes. Kit according to claim 11 for use in a test according to claim 2 or 3 or. one of claims 4 to 10 in dependence of claim 2 or 3 further comprising factor V depleted plasma. Kit according to claim 11 or 12, wherein at least one the reagents are in lyophilized form. A kit according to any one of claims 11 to 13, comprising exogenous reagent further comprising sodium chloride solution. The kit of claim 14, wherein the concentration of sodium chloride between 0.075 M and 0.15 M. Test according to one of claims 1 to 10 or kit according to one of the claims 11 to 15, wherein the exogenous reagent is between 0.01 and 0.2%, preferably 0.04 crude phospholipid. Test according to one of claims 1 to 10 and 16 or kit according to one of the claims 11 to 15, the exogenous reagent following the supernatant to the centrifugation of a 4% solution of acetone-dried Bovine brain is activated in saline solution. A test according to any one of claims 1 to 10, 16 and 17 or a kit according to any one of claims 11 to 15, wherein the concentration of PCA in the exogenous reagent with PCA is between 0.25 x 10 ^-8 M and 2.5 x 10 ^-8 M and preferably at 1.25 × 10 ^-8 M.